Enterococcus faecalis is a Gram-positive opportunistic pathogen and causative agent of urinary tract and wound infections, bacteremia, and infectious endocarditis. Like many other Gram-positive pathogens, enterococci carry mobile DNA elements in their genomes that mediate the spread of antibiotic resistance and other virulence determinants, complicating efforts by the medical community to control associated infections. Mobile DNA elements code for type IV secretion systems (T4SS's); these are multicomponent translocation channels responsible for delivery of DNA substrates to bacterial target cells. In many important Gram-negative pathogens, T4SS's are employed not only for interbacterial DNA transfer but also for injection of effector proteins into eukaryotic target cell. Upon translocation, the effectors disrupt host cellular processes allowing for growth and proliferation of the invading pathogen. Recent studies suggest that T4SS's of Gram-negative and -positive bacteria display conserved mechanisms of action in their ability to recognize and translocate cognate substrates across their cell envelopes. Thus, it is surprising that no studies have yet examined the potential for Gram-positive pathogens to use T4SS's for delivery of effector proteins to eukaryotic host cells during infection. Here, we propose to test a hypothesis that T4SS's of Gram-positive bacteria, like their Gram-negative counterparts, contribute to the infection process through delivery of effector proteins into eukaryotic target cells or the milieu. Two initial findings strongly support our hypothesis: i) we have gained evidence for protein translocation by the pCF10-encoded T4SS from E. faecalis donor cells to Escherichia coli recipients, and ii) a functional T4SS encoded by an 89-kilobase pathogenicity island (89K PAI) was shown to be necessary for virulence of another Gram-positive pathogen, Streptococcus suis. We will combine the expertise of the Christie and Dunny groups to identify protein substrates, secretion signals, and host range for protein translocation by two E. faecalis T4SS's, one encoded by the pheromone-responsive plasmid pCF10 and the second by the chromosomal 153-kb pathogenicity island (PAI). We will integrate the goal of defining the substrate repertoire of these Gram-positive T4SS's with infection assays using a rabbit endocarditis infection model to define the contribution of T4SS-mediated protein translocation to E. faecalis pathogenesis. We anticipate that our investigations will establish a proof-of-principle for the contribution of T4SS-mediated protein translocation to virulence of a Gram-positive pathogen. Such an outcome will stimulate new research initiatives aimed at deciphering mechanistic and cellular consequences of intercellular protein translocation during infection by E. faecalis and other medically-important Gram-positive pathogens.